Constructs

APache protein is encoded by the KIAA1107 gene. For detailed description of constructs used for APache silencing see Piccini et al. (2017) [25]. Briefly, shRNA targeting the 3’ untranslated region (UTR) of the mouse Kiaa1107 transcript and control shRNA (Luciferase shRNA; Sigma-Aldrich) were inserted into the pLKO.1-CMV-mCherry lentiviral vectors. For rescue experiments, the p277-EGFP-APache vector was used, being intrinsically resistant to APache-shRNA. For APache silencing through transfection, APache shRNA and control shRNA were inserted into pLKO.1-CMV-eGFP or pLKO.1-CMV-mTourquoise vectors. Expression plasmid encoding EGFP-APache was described previously (Piccini et al., 2017). mRFP-TrkB was a kind gift from Dr. Natalia Kononenko (CECAD, University of Cologne, Germany). AP-2µ2-mCherry was a gift from Christien Merrifield (Addgene plasmid # 27672; http://n2t.net/addgene:27672; RRID: Addgene 27672), pBABE-puro mCherry-EGFP-LC3B was a gift from Jayanta Debnath (Addgene plasmid # 22418; http://n2t.net/addgene:22418; RRID: Addgene_22418) and pTagRFP-C-LC3b (#FP141) was from Evrogen.

Transfection and transduction of primary neurons

Cultures of dissociated primary cortical neurons were prepared from embryonic 17/18-day (E17–18) brains of C57BL/6J mice (Charles River, Calco, Italy) and plated on poly-L-lysine (0.1 mg/ml, #25988-63-0, Sigma-Aldrich)-coated 25-mm glass coverslips at low density (6 × 104 cells/coverslip) and on poly-L-lysine (1 mg/ml)-coated 35-mm wells at high density (0.3–1 × 106 cells/well). Cells were maintained in a culture medium consisting of Neurobasal (#21103-049, Gibco) supplemented with B-27 (1:50 v/v, Gibco #17504-007, Gibco), Glutamax (1% w/v, #35050-38, Gibco), penicillin–streptomycin (1%, #10378-016, Gibco) and kept at 37 °C in a 5% CO2 humidified atmosphere. Under this culture condition approximately 85–90% of the cortical neurons are glutamatergic and cultures are almost glia-free [55]. All experiments were carried out in accordance with the guidelines established by the European Community Council (Directive 2010/63/EU of March 4, 2014) and were approved by the Italian Ministry of Health (authorization n. 600/2020-PR). For lentiviral infection experiments, neurons were transduced with 5 multiplicity of infection (MOI) of lentiviral vectors into cell medium at 12 days in vitro (DIV). After 24 h, half of the medium was replaced with fresh medium, and experiments were performed 5 days post infection. For rescue experiments, neurons were co-transduced with 5 + 5 MOI of lentiviral vectors. For plasmid and shRNA transfection experiments, neurons were transfected at 11–14 DIV using Lipofectamine™ 2000 (#11668019, Thermo Fisher Scientific) according to the manufacturer’s instructions and analyzed 3 days later. When specified, neurons were treated for 4 h at 37 °C with 250 nM Torin1 (#2273, BioVision) and/or 5 µg/ml cycloheximide (#C7698, Sigma-Aldrich), or an equivalent volume of DMSO/Milli-Q water as a solvent control.

Immunocytochemistry and fluorescence microscopy

Low-density primary cortical neurons were fixed at 17 DIV in 4% paraformaldehyde/4% sucrose in phosphate-buffered saline (PBS), pH 7.4 for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 min and blocked with 0.1% Triton X-100, 3% fetal bovine serum in PBS for 30 min. Samples were incubated with the following primary antibodies diluted in blocking solution for 2 h at room temperature or overnight at 4 °C: rabbit anti-APache (1:500, PRIMM EFA/10 201010-00019), rabbit anti-LAMP1 (1:200, #L1418, Sigma-Aldrich), rabbit anti-LC3B (1:200, #L7543, Sigma-Aldrich), mouse anti-LC3B (1:100, #AM20213PU-N, Origene), rabbit anti-p62/SQSTM1 (1:500, #P0067, Sigma-Aldrich), mouse anti-pan-axonal neurofilament marker (1:500, #SMI-312R, Covance), rabbit anti-Rab7 (1:250, #ab137029, Abcam), mouse anti-VAMP2 (1:500, #104211, Synaptic Systems), guinea pig anti-VAMP2 (1:500, #1042, Synaptic Systems). Immunostaining was detected using Alexa 488, and/or 647-Fluor-conjugated secondary antibodies (1:500, Thermo Fisher Scientific) diluted in blocking solution for 1 h at room temperature. Samples were mounted in Prolong Gold Antifade reagent with or without 4′,6′-diamidino-2- phenylindole (DAPI) (Thermo Fisher Scientific, respectively #P36934 and #P36935). Confocal images were acquired with a confocal laser-scanning microscope (SP8, Leica Microsystems GmbH, Wetzlar, Germany) with a 40X/1.4 oil-immersion objective. Each image consisted of a stack of images taken through the z-plane of the cell acquired every 300 nm. For each set of experiments, all images were acquired using the same optical acquisition settings for all conditions, the offset parameters in same cases were adjusted to better visualize and isolate APache and LC3 synaptic staining. Offline analysis was performed using ImageJ/FiJI and mean fluorescence intensities were quantified on ROIs manually selected on the cell soma or automatically detected using the “Analyze Particle” tool of ImageJ for synaptic boutons detection. VAMP2-positive puncta with areas of 0.1–2 µm2 were considered bona fide synaptic boutons. Co-localization studies were performed using the “JACoP” co-localization plug-in and calculating the Manders’ coefficient (shown in percent on the graph) in ROIs manually selected on the cell soma or at VAMP2-positive puncta using the ImageJ selection tool. For the analysis of LC3- or Rab7-positive puncta along neurites, a constant threshold was applied to all images and the number of fluorescent puncta was determined using the “Analyze particles” tool of ImageJ in manually selected ROIs (30-µm length). For the analysis of the mCherry-eGFP-LC3B tandem assay, the ratio of the mean fluorescence intensity of eGFP over mCherry was calculated at the cell soma. For quantitative analysis of the density of VAMP2-positive puncta, masks of mCherry-infected neurons were created using ImageJ/FiJI and the VAMP2 signal was thresholded using the same parameters for all the images. Colocalization points between the mCherry mask and VAMP2 signal, identified with the ImageJ/FIJI “JACoP” co-localization plug-in, represent VAMP2-positive bona fine synaptic puncta of infected neurons. The density of VAMP2-positive puncta was calculated as the number of puncta normalized on the mCherry neuronal area.

Live-cell neuron imaging and analysis

Low-density primary cortical neurons co-transfected with EGFP-APache and mRFP-LC3 or either control eGFP-shRNA (control) or eEGFP-APache-shRNA (APache KD) and mRFP-LC3b or mRFP-TrkB were imaged 3 days later in Tyrode’s solution (10 mM glucose, 140 mM NaCl, 2.4 mM KCl, 10 mM Hepes, 2 mM CaCl2, 1 mM MgCl2) in an environmental chamber at temperature-controlled stage (37 °C) on an inverted epifluorescence microscope (Olympus IX81, Olympus Corporation, Tokyo, Japan) with a 60X oil-immersion objective. Axons were identified based on morphological parameters [56]. Time-lapse recordings were acquired at 0.25–1 Hz for 30–120 s with Olympus xCellence rt Software. Frame rates were optimized for each experimental approach to capture events with high time resolution while minimizing photobleaching. Within each experiment, all conditions were imaged with the same paradigm. Motility of mRPF-LC3 or mRFP-TrkB puncta was analyzed by generating kymographs using “KymographBuilder” plug-in in ImageJ/FIJI. Vesicles were manually tracked with the “Manual Tracking” plug-in in ImageJ/FIJI. Retrograde and anterograde motile mRFP-LC3b or mRFP-TrkB puncta were identified by the respective location of the cell soma or axonal tip during imaging. The retrograde mean speed of individual fluorescent puncta was calculated by determining the net displacement over the time-lapse. Stationary puncta were defined as those moving at a velocity < 0.05 μm/s over the imaging period.

Transmission electron microscopy (TEM)

Low-density cultures of cortical neurons were infected at 12 DIV with either control shRNA or APache shRNAs plus APache-EGFP in rescue experiments and processed for TEM. Neurons were fixed at 17 DIV with 1.2% glutaraldehyde in 66 mM sodium cacodylate buffer, post-fixed in 1% OsO4, 1.5% K4Fe(CN)6, 0.1 M sodium cacodylate, en bloc stained with 10% of uranyl acetate replacement stain (EMS) for 30 min, dehydrated, and flat embedded in epoxy resin (Epon 812, TAAB). After baking for 48 h, the glass coverslip was removed from the Epon block by thermal shock and neurons were identified by means of a stereomicroscope. Embedded neurons were then excised from the block and mounted on a cured Epon block for sectioning using an EM UC6 ultramicrotome (Leica Microsystems). Ultrathin Sects. (60–70 nm thick) were collected on 200-mesh copper grids (EMS) and observed with a JEM-1011 electron microscope (Jeol, Tokyo, Japan) operating at 100 kV using an ORIUS SC1000 CCD camera (Gatan, Pleasanton, CA). For each experimental condition, at least 50 images of synapses and neurites were acquired at 10,000x magnification (sampled area per experimental condition: 36 µm2). Synaptic profile area, AV number and density were determined using ImageJ. AVs were defined as single or double membrane-bound vacuoles containing intracellular material.

Western blot analysis

Total cell lysates from high-density cortical neuronal cultures at 17 DIV were extracted in lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA pH 8, 1% Triton X-100) supplemented with protease and phosphatase inhibitors cocktail (respectively #58715 and #58705, Cell Signaling). After 10 min of incubation on ice, lysates were collected and clarified by centrifugation (10 min at 1,000 x g at 4 °C). For digitonin-based membrane/organelle-enriched and cytosol fractionation, neurons were washed in HBSS and permeabilized in buffer D (modified from 57): 0.02% Digitonin (#D-5628, Sigma-Aldrich), 300 mM sucrose, 5 mM Hepes, 100 mM NaCl, 5 mM EDTA, 3 mM MgCl2 supplemented with protease inhibitor cocktail under gentle shaking at 4 °C for 20 min. Supernatant (cytosolic fraction) was collected, and membranes were scraped from the well plate in buffer D. Protein concentration was determined using either Bradford Protein Assay (#5000006, Bio-Rad) or Pierce™ BCA Protein Assay Kits (#23227, Thermo Fisher Scientific) assay, and equivalent amounts of protein were subjected to SDS-PAGE on polyacrylamide gels (8-14%) and blotted onto nitrocellulose membranes (Whatman). Blotted membranes were blocked for 1 h in 5% milk in Tris-buffered saline (10 mM Tris, 150 mM NaCl, pH 8.0) plus 0.1% Triton X-100 and incubated 2 h at room temperature or overnight at 4 °C with the following primary antibodies: mouse anti-actin (1:1000, #A4700, Sigma-Aldrich), rabbit anti-APache (1:1000, PRIMM EFA/10 201010-00019), mouse anti-AP-2α (1:1000, #610502, BD Transduction Laboratories), rabbit anti-AP2β (1:2000, #NBP2-15563, Novus Biologicals), mouse anti-AP2µ (1:1000, #611350, BD Transduction Laboratories), rabbit anti-ATG3 (1:1000, #3415P, Cell Signaling), rabbit anti-ATG5 (1:1000, #8540P, Cell Signaling), rabbit anti-ATP6V1A (1:1000, #ab137574, Abcam), rabbit anti-caspase-3 (1:1000; #9662, Cell Signaling), rabbit anti-cathepsin D (1:2000, #219361, Millipore), rabbit anti-EGFR (1:1000, #ab52894, Abcam), rabbit anti-GAPDH (1:5000, #2118, Cell Signaling), rabbit anti-LAMP1 (1:1000, #ab24170, Abcam), rabbit anti-LC3B (1:1000, #L7543, Sigma-Aldrich), rabbit anti-mTOR (1:1000, #2983, Cell Signaling), rabbit anti-phospho-mTOR Ser2448 (1:1000, #5536, Cell Signaling), mouse anti-NaK3 (ATP1A3) (1:1000, #MA3-915, Invitrogen), mouse anti p150/Glue (1:1000, #610474, BD Transduction Laboratories), rabbit anti-Rab5 (1:1000, #ab18211, Abcam), mouse anti-Rab7 (1:1000, #ab50533, Abcam), mouse anti-synaptophysin (anti-SYP; 1:5000, #101011, Synaptic Systems), mouse anti-synaptotagmin1 (anti-Syt1; 1:1000, #105011, Synaptic System), rabbit anti-ULK1 (1:1000, #8054, Cell Signaling), rabbit anti-phospho-ULK1 Ser757 (1:1000, #6888, Cell Signaling), mouse anti-vinculin (1:500, #V9264, Sigma-Aldrich). Membranes were washed and incubated for 1 h at room temperature with peroxidase-conjugated goat anti-mouse ((#1706516, 1:3000; Bio-Rad) or anti-rabbit (#1706515, 1:5000; Bio-Rad) secondary antibodies. Immunoreactivity was detected using the ECL chemiluminescent detection system (#32106, Thermo Fisher Scientific) and autoradiography or by ChemiDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). Quantification was performed by bands densitometric analysis with ImageJ/FIJI for Windows (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA). For quantification, protein levels were always first normalized to the loading control from each corresponding lane and then the levels in the treated/APache KD neurons were normalized relative to controls set to 100%.

Immunoprecipitation assay

Mouse brain was homogenized in ice-cold buffered sucrose solution (0.32 M sucrose, 5 mM HEPES, pH 7.4) plus 100 mM NaCl and protease inhibitors and cleared by low-speed centrifugation (1,000 × g for 10 min at 4 °C). The supernatant was incubated with 1% Triton X-100 for 30 min at 4 °C and then centrifuged at 150,000 x g for 45 min at 4 °C. Equivalent amounts of brain extract were incubated for 3 h at 4 °C with either rabbit anti-APache or rabbit control IgG (10 µg/sample) pre-coated overnight with Protein G-Sepharose (GE Healthcare). After extensive washes, samples were resolved by SDS–PAGE and analyzed by Western blotting.

Cathepsin D activity assay

Cellular Cathepsin D (CTSD) activity was determined using a CTSD activity assay kit (#K143, BioVision), according to manufacturer’s protocol. The assay is based on the use of the fluorescently labeled preferred CTSD substrate sequence GKPILFFRLK(Dnp)-DR-NH2 that emits fluorescence after cleavage. Briefly, transduced primary cortical neurons were lysed at 17 DIV in 200 µl of chilled CD Cell Lysis Buffer, incubated on ice for 10 min, and centrifuged for 5 min at top speed. Ten µg /well of cell lysate were mixed with 50 µl of Reaction Buffer and 2 µl of substrate to a final volume of 102 µl into a 96-well plate and incubated for 2 h at 37 °C. Samples were read in triplicate at 10-min intervals (Ex/Em = 328/393 nm) at multiplate TECAN® reader (Tecan Trading AG, Switzerland) using a 320 ± 25-nm excitation filter and 485 ± 20-nm emission filter. Background values were calculated by reading wells filled only with solutions and subtracted to each sample value. Data were normalized to µg protein/sample and expressed in percentage of control value.

Epidermal growth factor receptor degradation assay

Epidermal growth factor receptor (EGFR) degradation assay was performed as previously described [58] with minor modifications. High-density cortical neurons at 17 DIV were incubated with 200 ng/ml EGF (#PHG0313, Thermo Fisher Scientific) for 15 min at 4 °C allowing ligand-receptor binding. To synchronize ligand-receptor internalization, the ligand-containing medium was replaced by fresh prewarmed medium. Cells were incubated at 37 °C allowing ligand-receptor internalization for 0, 1 and 4 h in the presence of 5 µg/ml cycloheximide to inhibit the de novo synthesis of EGFR. Total cell lysates were analyzed by Western blot using an anti-EGFR antibody to monitor degradation of EGFR band.

Human brain tissue

We used previously characterized [39] frozen blocks and formalin-fixed autopsy sections of the frontal cerebral cortex from 8 cases with late-onset sporadic Alzheimer’s disease (AD) (mean age at death 80 ± 8 years; post-mortem interval 8 h ± 3; clinical history of disease; pathological diagnosis according to the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) criteria) provided by the brain bank of Case Western Reserve University, Cleveland, OH, and from 8 cognitively normally aging elderly subjects as controls (mean age at death 83.3 ± 10 years, post-mortem interval 9.5 h ± 4). The latter subjects had been tested neuropsychologically annually and agreed to be autopsied for research purposes (provided by the Alzheimer’s Disease Research Center, University of Kentucky). Their neuropsychological scores were within the normal range; in the cerebral cortex abundant amyloid plaques were present with absent or scarce neurofibrillary pathology. Protein lysates from autopsy sections were extracted in ice-cold buffered sucrose solution (0.32 M sucrose, 5 mM Hepes, pH 7.4) supplemented with 100 mM NaCl and protease inhibitors cocktail and centrifuged at 1,000 x g for 10 min at 4 °C to obtain a post-nuclear supernatant fraction.

Statistical analysis

Normal distribution of experimental data was assessed using the D’Agostino-Pearson’s normality test (n > 6) or Shapiro-Wilk test (n ≤ 6). To compare two normally distributed sample groups, the unpaired Student’s t-test with the Welch’s correction was used. To compare two sample groups that were not normally distributed, the nonparametric Mann–Whitney’s U-test was used. To compare more than two normally distributed sample groups, we used one- or two-way ANOVA followed by the Bonferroni’s multiple comparison test. In cases in which more than two sample groups were not normally distributed, we used the Kruskal–Wallis’s ANOVA test followed by the Dunn’s multiple comparison test. Statistical analysis was carried out using Prism 7.0 software (Graphpad Software Inc., La Jolla, CA). Significance level was preset to p < 0.05. Data are expressed as means ± standard error of the mean (SEM) for number of cells/samples or independent preparations (n) as detailed in the figure legends.